Understanding the biological mechanisms of nerve regeneration and degeneration is an important step towards the development of novel therapies for human neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and other neurological disorders affecting millions of people. The regeneration processes can be studied by severing an axon in a controlled manner and then observing its re-growth and functional recovery. To sever an axon in-vivo, a high precision and non-intrusive cutting tool is required. In the absence of precision techniques for severing axons (axotomy), investigations have so far been limited to complex organisms (mouse and zebrafish). Just recently we demonstrated that femtosecond laser pulses can be used for axotomy in the roundworm Caenorhabditis elegans (C. elegans) and those axons functionally can regenerate after the operation. The advantage of these ultrashort laser pulses is their ability to evaporate an extremely small volume of tissue without heating or damaging the surrounding cells. Application of this precise surgical technique now enables nerve regeneration to be studied in-vivo in organisms with simple nervous systems. Since simple organisms such as C. elegans have amenable genetics, application of the femtosecond laser axotomy technique will help in the rapid identification of genes and molecules that affect nerve regeneration and degeneration. The goal of this research project is to develop a high-throughput laser nano-surgery platform for axon regeneration & degeneration studies in C. elegans in-vivo. Specifically, an integrated microfluidic device will be developed to trap the worms. This microfluidic trap will facilitate the appropriate immobilization of the animals for precision nanosurgery of axons using femtosecond laser pulses and for imaging the re-growth of the injured axons following nanosurgery. Development of a high throughput screening platform requires integration of different modules for nanosurgery, feeding, and imaging and their synchronization through computer controlled automation. A platform having 100's of chambers for feeding of individual worms will facilitate automated surgical and screening studies of multiple worms, thus greatly reducing time and cost. For successful development of a high-throughout nanosurgery platform for in-vivo nerve regeneration/degeneration studies, we have assembled a multi-disciplinary team with expertise in three critical areas (1) ultrafast laser ablation of bio-materials (Dr. Ben-Yakar), (2) fabrication and assembly of microfluidic systems (Dr. Chronis), and (3) developmental neurobiology of C. elegans (Dr. Bargmann). Concurrently with the development of a high-throughput microfluidic platform, two important nerve regeneration/degeneration problems will be investigated: (1) time-lapse in-vivo imaging of axon re-growth and degeneration of injured axons and (2) the role of the Sir2 family of genes and the drug resveratrol in regeneration of injured axons. Understanding the biological mechanisms of nerve regeneration and degeneration is an important step towards the development of novel therapies for human neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and other neurological disorders affecting millions of people. Development of a high-throughput laser nano-surgery platform applicable to simple organism such as C. elegans in-vivo will help in the rapid identification of genes and molecules that affect nerve regeneration and degeneration. [unreadable] [unreadable] [unreadable]